1. Field of the Invention
The field of the invention relates to high throughput screening methods to provide specific probes that measure levels of unbound analytes, including unbound free fatty acids and other unbound metabolites. Also disclosed are probes obtained with the high throughput screening methods and the use of a combination of probes to determine an unbound free fatty acid profile or more generally an unbound metabolite profile for an individual.
2. Description of the Related Art
For purposes of the present disclosure, fatty acids are non esterified carboxylated alkyl chains of 1-30 carbons atoms which may exist as neutral (e.g. protonated, sodium or potassium salt) or ionic species, depending upon the pH and conditions of the aqueous media. Free fatty acids (FFA) are equivalent to fatty acids and both terms refer to the totality of FFA including those in aqueous solution as monomers plus those that are not in solution (for example bound to other macromolecules (proteins, membranes), cells or part of an aggregate of FFA (micelles, soaps and other more complex aggregates). FFA present as monomers in aqueous solution (either charged or neutral) are referred to as unbound free fatty acids (FFAu). For the purposes of the present disclosure, probes are fluorescently labeled proteins that upon binding an analyte, such as a FFAu, reveal a measurable change in fluorescence.
For purposes of the present disclosure, metabolites are physiologically important molecules whose molecular weight is approximately 2000 Da or less. These include molecules that occur naturally in the course of human or animal physiology or pathophysiology, and drug molecules and their metabolic products and nutrient molecules and their metabolic products. Similar to FFA and depending upon their solubility, a fraction of each metabolite is present as monomers in aqueous solution (either charged or neutral). We refer to this fraction as the unbound metabolite. For the purposes of the present disclosure, probes are fluorescently labeled proteins that reveal a measurable change in fluorescence upon binding to unbound metabolite.
For the purposes of the present disclosure, the term “lipid” is taken to have its usual and customary meaning and defines a chemical compound which is most soluble in an organic solvent but has some level of solubility in the aqueous phase (the fraction that is unbound). Accordingly, a “lipid-binding protein” includes any protein capable of binding a lipid as lipid is defined herein.
Levels of unbound molecules, such as for example lipids, hormones and metabolic products, can provide information diagnostic of health and disease when measured in appropriate human or animal fluids. It is increasingly apparent that determination of the unbound (a.k.a ‘aqueous phase’ or ‘free’) concentration of such molecules provides critical information about physiologic homeostasis. Many metabolites are hydrophobic molecules with low aqueous solubility and unbound concentrations that are much lower than their “total” concentration, where the bulk of the “total” may be bound to proteins or cells.
Intracellular lipid binding proteins (iLBP) are a family of low-molecular weight single chain polypeptides. There are four recognized subfamilies. Subfamily I contains proteins specific for vitamin A derivatives such as retinoic acid and retinol. Subfamily II contains proteins with specificities for bile acids, eiconsanoids, and heme. Subfamily III contains intestinal type fatty acid binding proteins (FABPs) and Subfamily IV contains all other types of fatty acid binding protein (Haunerland, et al. (2004) Progress in Lipid Research vol. 43: 328-349). The entire family is characterized by a common 3-dimensional fold. Ligand binding properties of the different subfamilies overlap considerably. The wild type proteins of subfamilies I (Richieri et al (2000) Biochemistry 39:7197-7204) and II both bind fatty acids and those of subfamily II bind fatty acids as well as their native ligands. Moreover, single amino acid substitutions are able to interconvert the ligand binding properties of proteins of subfamilies I and II (Jakoby et al (1993) Biochemistry 32:872-878).
U.S. Pat. Nos. 5,470,714 and 6,444,432, which are incorporated herein by reference, describe probes for the determination of unbound free fatty acids (FFAu). These probes were constructed using either native or mutant forms of proteins from the iLBP family. As discussed above, this family includes FABPs (Banaszak et al (1994) Adv. Protein Chem. 45:89-151; Bernlohr et al (1997) Ann. Rev. Nutrition, 17: 277-303). FABPs are intracellular proteins of approximately 15 kDa molecular weight and have a binding site that binds 1 or 2 FFA. Unfortunately, there is currently no way to determine the concentrations of different FFAu in mixtures of FFAu. Similarly, there are no general methods for determining the unbound concentrations of other important metabolites such as, for example other lipids, hormones, and drugs. This is largely due to the low concentration at which these components are present and their often poor solubility properties in aqueous solutions.
Unfortunately, despite the availability of protein structures and co-complex structures with ligands of interest, existing state of the art of molecular theory is not sufficient to design probes with the desired specificity and sensitivity de novo. Thus, extensive experimentation is typically required to find protein probes that not only bind with the desired specificity, but also produce a measurable signal indicative of ligand binding. Improving specificity and signaling through a completely random mutational strategy is not practical even for a small protein such as an FABP because a) there are 20131 possible mutants for a 131 residue FABP, and b) testing even a single probe using established state of the art methods requires extensive time (at least 2 weeks/probe) for purification, reaction chemistry and probe fluorescence response characterization. Even if a more modest library of mutants is generated through random mutagenesis in specific regions of the protein, a method is needed to rapidly generate and screen the thousands of resulting mutant probes. Each mutant needs to be produced, and chemically reacted with a fluorescent group, in sufficient quantity to enable the measurement of its sensitivity and selectivity for many different ligands. It is also critical that the probes be as free as possible of contaminating proteins, unreacted fluorophore, and any other compounds that might interfere with sensitive fluorescence measurements. The development of a rapid, automated method for measuring and comparing probe responses to ligand is also critical. Embodiments of the invention described here satisfy these needs by disclosing “high throughput” methods for the rapid a) generation of large numbers of probes and the b) screening and characterization of these probes. An important aspect of this invention is that it allows the previous necessary and very time consuming step of characterization of ligand binding to the protein to be omitted; only the probe itself is characterized. This is important not only for the avoidance of the protein characterization step but also because the properties of the probe are often not predictable from the ligand-protein binding characteristics. For example, different proteins can have very similar binding affinities but the fluorescence response of their derivative probes can be quite different.